Project Abstract Obesity has multiple attendant metabolic comorbidities, including Non-Alcoholic Fatty Liver Disease (NAFLD). NAFLD is now the leading cause of chronic liver disease, with prevalence approaching 30% in certain populations, but may in fact be considered a ?pre-disease? state for Non-Alcoholic Steatohepatitis (NASH). NASH, defined by hepatocyte damage with associated inflammation and fibrosis, predisposes to cirrhosis and hepatocellular cancer, and is the fastest-growing reason for liver transplantation. NASH has no approved pharmacotherapy ? as the prevalence of obesity-related NASH continues to rise, and available livers for transplantation remain limiting, this unmet need grows more urgent. Notch is a highly conserved family of proteins critical for cell fate decision-making, but less is known about Notch action in mature tissue. Our initial characterization demonstrated that Notch activity is present at low levels in normal liver, increases markedly in livers from obese patients and diet-induced or genetic mouse models of obesity, but is highest in patients with NASH and shows significant positive correlations with plasma ALT and NAFLD Activity Score. This observational study prompted a pilot sub-study of the PIVENS trial, where we observed that improved NASH is associated with lower hepatic Notch activity. We next found that Notch loss-of-function mice are protected from diet-induced steatohepatitis and fibrosis, whereas constitutively-active hepatocyte Notch activity prompted inflammation and fibrosis even in chow-fed mice. Intriguingly, in NASH patients and mouse models of steatohepatitis, expression of only 1 of 5 Notch ligands (Jag1) correlates with disease pathology. These results suggest that Jag1-driven Notch activity in liver may be more than a biomarker, but actually a mechanistic determinant of NASH/fibrosis, which we will test in this application. In Aim 1, we study mechanistic determinants of increased Jag1 in the obese, inflamed liver, determine whether hepatocyte Jag1 is necessary for increased liver Notch signaling in steatohepatitis and test whether novel Jag1 inhibitors may reduce obesity-induced liver inflammation and fibrosis. In Aim 2, we will test whether changes in hepatocyte secretion of MCP1 and Osteopontin, both identified using an unbiased screen for markers of Notch-active hepatocytes in mice with NASH, can explain Notch-mediated hepatic inflammation and fibrosis respectively, as well as mechanism underlying Notch-induced transcription of these molecular mediators. Achieving the goals of this application will identify the mechanism of increased hepatocyte Notch signaling in the obese liver, delineate the mechanistic determinants of Notch-induced liver pathology and potentially lead to application of novel Notch inhibitors for NASH and fibrosis.